Dual Intracellular and Extracellular Expression Technology (dIEE) for Alzheimer's Disease Abstract Multiple intertwined mechanisms contribute to the pathogenesis of Alzheimer's Disease (AD), a devastating neurodegenerative illness afflicting over 5 million Americans. Aggregates of the microtubule-associated protein tau represent a major pathological finding in AD and other neurodegenerative tauopathies and unlike extracellular A?-amyloid plaques, correlate well with progressive cognitive decline. Exposure of the N-terminal phosphatase-activating domain (PAD) may be one of the earliest and most consequential conformational changes of tau. Intracellular microinjection of recombinant tau near the synapse exposes PAD, and a peptide corresponding to the PAD itself leads to synaptic dysfunction. While tau is normally localized intracellularly, during AD a positive feedback loop with A? can spread damage extracellularly throughout the brain. Pathological tau then creates two challenges for a potential therapeutic: a) block intracellular initiation-activity and b) clear toxic extracellular tau to prevent progression. To address these problems, we have designed a recombinant adeno-associated viral (AAV) vector to achieve long-term, stable central nervous system (CNS) delivery of an antibody. This vector will simultaneously deliver an intracellular anti-tau PAD single-chain intrabody (iAb) combined with a secreted extracellular anti-Tau N-Terminal (TNT) antibody (sAb). Our novel dual intrabody/antibody approach employs proprietary dual Intracellular and Extracellular Expression technology (dIEE). We hypothesize that the combined effect of the anti-tau intrabody and secreted antibody will reduce tau-mediated pathology with concomitant improvement of AD. In addition, AAV-delivery addresses the blood brain barrier (BBB) problem of systemic antibody therapy and represents a novel and potentially practical approach for the treatment of additional neurodegenerative diseases. In Phase I, we will construct the vectors, and test their expression of TNT1-iAb and TNT1-sAb in vitro and in the brains of recipient mice. We are optimistic that our AAV dual expression system for delivery of anti-tau-PAD domain intrabodies and antibodies to the brains of affected patients will provide a novel restorative therapy for AD.